Commercially obtained cystine binding protein (CBP) of E. coli has been purified by HPLC, yielding a pure cystine binding protein not similar to any protein in the data banks. This protein has been partially sequenced, an N-terminal peptide made and an antibody to this peptide obtained which reacts with the whole protein. The commercial preparation of cystine binding protein also contains a histidine binding protein which can be used to assay for histidine in the same way that cystine binding protein is used to assay tissues for cystine. Cystine binding protein of E. coli is being cloned by two methods. One, by using an E. coli expression library and identifying the correct protein producing colony with our antibody to cystine binding protein. Two, by making oligonucleotides of sections of the known N-terminal sequence of cystine binding protein and using them as primers to identify this protein in the E. coli library. Niemann-Pick C disease was shown to be reversibly induced in normal fibroblasts by hydrophobic amines and certain steroids, thus creating a model system for study of this disease. We are identifying the trafficking patterns and metabolism of intracellular cholesterol using various inhibitors and stimulators. We have shown that normal and cystinotic fibroblasts take up ascorbic acid by two mechanisms, a high affinity Na+ dependent transporter and a low affinity Na+ independent transporter, neither of which can be accounted for by diffusion. Transport of ascorbic acid by the low affinity transporter is not different from normal, however transport in cystinotic cells by the high affinity transporter is about 50% of that seen in normal cells.